Cytotoxicity of leaves, stems, and flowers of Kecubung ( Datura metel ) extracts using the Brine Shrimp Lethality Test (BSLT) method

Brine shrimp ( Artemia salina ) cytotoxicity assays (BSLT) are one of the commonly used cytotoxic test methods to assess a plant extract's pharmacological activity and toxicity. Thus, this study aimed to examine the toxic levels of Kecubung ( Datura metel ) leaves extracted using different solvents, namely ethanol and ethyl acetate. The results showed that the LC 50 value of flower ethanol, flower ethyl acetate, leaf ethanol, and stem ethyl acetate extracts had an LC 50 value <1000, which was included in the toxic category. The LC 50 value of flower ethanol is 121.044 ppm, flower ethyl acetate 105.89 ppm, leaf ethanol 639.589 ppm, and stem ethyl acetate 635.276 ppm. Ethanol leaf extract at a concentration of 1000 ppm showed the highest mortality with a percentage of 60% of the total number of A. salina . The flower ethanol extract showed the highest mortality at a concentration of 250 ppm with a percentage of 67% and at a concentration of 1000 ppm with a percentage of 70% of the total number of A. salina . Meanwhile, flower ethyl acetate and stem ethyl acetate extract at a concentration of 1000 ppm obtained 100% mortality of A. salina in the first 6 hours. The result shows that the flower ethyl acetate and stem ethyl acetate extract at a concentration of 1000 ppm is very toxic compared to other concentrations.


Introduction
Kecubung (Datura metel) is one of the wild plants that can be used as herbal medicine which is widespread in lowland areas up to an altitude of 800 meters above sea level.All parts of the D. metel plant have active compounds consisting of roots, stems, leaves, flowers, and fruits.Behind these active compounds, D. metel also has benefits as traditional medicines and has been used as an anti-bacterial, antiseptic, narcotic, and sedative for centuries (Ganesh et al., 2015).
Bioactive compounds and alkaloids in the D. metel included fatty substances, steroids, phenolic saponins, tannins, and tropane alkaloids: such as atropine, hyoscyamine, scopolamine, hyoscine, metosdina, norhiosiamina, norscopolamine, cuschohygrine, and nicotine (Huong, 1990;Thomas, 2003).The high content of alkaloids in the D. metel can be used as a natural pesticide for pest control in the fish ponds.In addition, alkaloids can also be used in fish as an anesthetic in the transportation processes.The goal is to reduce stress levels and fish deaths on the transportation processes.Natural anesthetic from D. metel with the compounds contained can give the fish unconscious or fainting effect.
Toxicity is the ability of a substance that has toxic properties, so that it can cause organ damage to organism.Rahmawati and Romi (2017) stated that toxicity is an effect that causes functional, Depik Jurnal Ilmu-Ilmu Perairan, Pesisir dan Perikanan Volume 12, Number 2, Page 117-124 Humairani et al. (2023) biochemical, or physiological (structural) disorders that can cause pain that interferes with the health of the organism's body.The tropane alkaloids contained in the D. metel plant are anticholinergic alkaloids that can be toxic to the nervous system, so that the safety limit for their use needs to be set (Sharma et al., 2021).
Based on this description, the researcher aims to calculate the effect of D. metel toxicity on Artemia salina by using the BSLT (Brine Shrimp Lethality Test) method.In addition, the purpose of this study was to calculate and analyze the toxicity levels and compare the toxicity levels of stem, leaf, and flower extracts.BSLT is a toxicity test method using one of the aquatic animals, in the form of A. salina larvae (Meyer et al., 1982).This method is most commonly used because of the easy, fast and low cost of treatment.
Several studies have shown that BSLT can be used to measure the toxicity of herbal materials for medical purposes.BSLT has also been used to measure the toxicity of herbal compounds that have the potential to be used as anesthetic ingredients (Purbosari et al. 2022).The BSLT test is generally used to see 50% mortality of test animals exposed to herbal extracts or the effectiveness of the concentration of compounds that cause toxicity in test animals.However, the use of the BSLT method can only be used to determine the initial concentration to be used for the test because the toxicity effect is not able to describe physiological damage due to compound toxicity (Setiani et al. 2023).

Location and time of research
This research is an experimental study with white flowered D. metel plant extract with an extraction solution using ethanol and ethyl acetate.The plant parts of D. metel used include; stems, leaves and flowers.The method used in the toxicity test is the Brine Shrimp Lethality Test (BSLT) method based on the method of Mayer et al. (1982).This method uses A. salina larvae as an organism toxicity test.This research was conducted from December 2021 to February 2022.All stages of extraction and anesthesia testing were conducted at the Aquaculture Laboratory, Faculty of Agriculture, Almuslim University, Bireun Regency, Indonesia.

Materials and tools preparation
The materials used in this study were D. metel, sea water, fresh water, A. salina, yeast, ethyl acetate and 90% ethanol.The tools used in this research are aquarium, blender, measuring cup, beaker, filter paper, test tube, soxhlet, aquarium, and aerator.
D. metel plants used as samples consisted of stems, leaves, and flowers obtained in the districts of Aceh Tengah and Bener Meriah.D. metel collected according to the part of the plant, then cleaned of dirt and aphids by washing under running water until clean.Then drained, then chopped into small pieces.Dry by airing in a room until completely dry.D. metel has been dried is mashed by means of a blender to obtain D. metel powder.

Preparation of D. metel extract
In this extraction step of D. metel using maceration method with two treatment solutions of the compound, namely ethyl acetate and 90% ethanol.This extraction has been modified from the study of Zulfahmi et al. (2018).D. metel powder was weighed according to the dose and put into a macerator then soaked with a solution of the compound used in the treatment (ethyl acetate or 90% ethanol), then covered with aluminum foil for 48 hours while stirring occasionally.The maceration results were filtered using filter paper (Whatman no.1).The results of the D. metel filter were weighed, then mixed with the solution (ethyl acetate or 90% ethanol) into one.The result of the filtration was evaporated with Soxhlet, so that a clear thick colored D. metel extract was obtained.The maceration process can be repeated twice using the same solvent.The results of the D. metel extract were weighed and ready for testing.

Identification of active compounds
GC-MS analysis was carried out to identify the active compounds contained in each extract.GC-MS analysis was performed using a Shimadzu GC-MS-QP2010 Ultra equipped with a 30-m × 0.25-mm × 0.25-μm Rxi-1MS column (Restek), and the initial temperature of the 100 o C column was heated for 5 minutes, then the temperature was gradually increased up to 250 o C at a rate of 10 o C min -1 .The split injector and the GC-MS interface are each at a temperature of 250 o C. The detectors used were massselective and electron-impact mass ionization spectrometry programmed at 70 eV and a temperature of 250°C.The carrier gas used was helium with a flow rate of 2.0 mL min −1 , and an injection volume of 2 L. Data was recorded using GC-MS Solution Software (Shimadzu).The components extract detected compared to the activity of the components with potentiality as anaesthetic agents in the literature existed.

Preparation of A. salina larvae
Artemia eggs are incubated in hatching funnels that have been filled with seawater and carried out under light.At the time of hatching, water quality  Humairani et al. (2023) parameters that must be considered are temperature, pH, salinity 28-30 ppt, and DO.

Toxicity test
Ten larvae of A. salina were transferred to each treatment tank using a 9-inch disposable pipette, and seawater was added to reach 5 mL.A drop of dry yeast suspension was added as feed to each tank.The treatment tank is placed under the light.Surviving A. salina was counted with the help of a 3x magnifying glass lens after 6 hours and 24 hours.If a control death occurred, the data were corrected by the Ordaz-Silva et al. (2016) formula, as follows: Determination of LC50 LC50 at doses of 100 ppm, 250 ppm, 500 ppm and 1000 ppm with 95% confidence intervals determined from a 24-hour count using the probit analysis method.If the data is not sufficient to perform this technique, then the dose-response data is transformed into a straight line using the logit transformation.The LC50 value is derived from the best line obtained from the regression analysis.

Identification of active compounds
GC-MS analysis data on ethanol extracts of leaves and flowers can be seen in Table 1, while ethyl acetate extracts of stems and flowers of amethyst plants, can be seen in Table 2.
Secondary metabolic compounds were identified for ethanol extracts only on the leaves and flowers.In the stem part, the yield produced is very small so it does not reach the minimum number of samples needed for GC-MS testing.A total of 19 compounds were identified in the leaves and 13 compounds in the flowers.In the leaves, the main components identified in the extract were hexadecanoic acid, ethyl ester (CAS) ethyl ester (15.37%), phytol (34.38%) and elaidic acid, (E)-9-octadecenoic acid ethyl ester (14.49%).In flowers, the main components identified were hexadecanoic acid, ethyl ester (CAS) ethyl ester (34.39 %) and elaidic acid, (E)-9octadecenoic acid ethyl ester (30.88 %).Identification of secondary metabolite compounds in ethyl acetate extract was carried out on the stem and flower of amethyst.In the leaves, the yield produced is also very small, so it does not reach the minimum number of samples needed for GCMS testing.The total compounds identified were 35 in the stem and 31 in the flower.The main compound components identified in the stem are Seychellene (7.51%), Pyrimidine, 2,4-dihydrazino-5-nitro-6-meth (8.10%), Hexadecanoic acid, methyl ester (10.74%).While the main compound components identified in the flower are 1,2,3-Propanetriol, monoacetate (18.44%) and Seychellene (7.46%).

Determination of LC50
Figure 1 shows that in the control, A. salina can live up to 100% without the addition of any extract.In contrast to the test tank which has been added to the ethanolic leaf extract at concentrations of 100 ppm, 250 ppm, 500 ppm and 1000 ppm, the survival rates of A. salina are 78%, 66%, 58% and 38%, respectively.Furthermore, leaf ethyl acetate extract at concentrations of 100 ppm, 250 ppm, 500 ppm and 1000 ppm showed the survival rates of A. salina reached 74%, 66%, 62% and 55%, respectively.
In the control, A. salina can live up to 100% without the addition of any extract (Figure 2).In contrast to the test tank which has been added to the ethanolic stem extract at concentrations of 100 ppm, 250 ppm, 500 ppm and 1000 ppm, the survival rates of A. salina are 72%, 72%, 76% and 56%, respectively.Furthermore, stem ethyl acetate extract at concentrations of 100 ppm, 250 ppm, 500 ppm and 1000 ppm showed the survival rates of A. salina reached 68%, 68%, 66% and 0%, respectively.
A. salina can live up to 100% without the addition of any extract in the control (Figure 3).In contrast to the test tank which has been added to the ethanolic flower extract at concentrations of 100 ppm, 250 ppm, 500 ppm and 1000 ppm, the survival rates of A. salina are 56%, 32%, 46% and 30%, respectively.Furthermore, flower ethyl acetate extract at concentrations of 100 ppm, 250 ppm, 500 ppm and 1000 ppm showed the survival rates of A. salina reached 30%, 36%, 30% and 0%, respectively.

Identification of active compounds
Both hexadecanoic acid, ethyl ester (CAS) ethyl ester and elaidic acid, (E)-9-octadecenoic acid ethyl found as main components in the ethanol extracts are fatty acid compounds.It is known to have biological activities such as antitumoral, antimicrobial, antioxidant, decrease blood cholesterol, antiinflammatory, hypocholesterolemic nematicide, pesticide, antiandrogenic flavour, hemolytic, 5-Alpha reductase inhibitor (Isbilen and Volkan, 2021).While phytol is a diterpene compound known to have biological activities as antimicrobial, cytotoxic, antioxidant, anticancer, apoptosis induction and autophagic protection, anxiolytic and anticonvulsant, immune-modulating, antinociceptive and antiinflammatory properties (Islam et al. 2018).
As mentioned above, the ethyl acetate extracts main components were Seychellene, Pyrimidine, 2,4dihydrazino-5-nitro-6-meth, Hexadecanoic acid, methyl ester and 1,2,3-Propanetriol, monoacetate.It is known that Seychellene is one of the components of patchouli oil and is the dominant component of Valeria celtica (Bicchi et al., 1983), Pogestemon cablin (Swamy and Sinniah, 2015) and also identified in Waldheimia glabra (Giorgi et al., 2013).Seychellene is known to have the ability as a non-selective inhibitor of cyclooxygenase, an enzyme that plays a role in the production of prostaglandins which are important mediators of pain and inflammatory responses  (Raharjo et al. 2017).While Propanetriol monoacetate was confirmed to be present in other plants exhibiting antimicrobial, anti-inflammatory, diuretic and anticancer effects (Foo et al., (2015).

Determination of LC50
Based on the results of the study, the LC50 value of flower ethanol extract, flower ethyl acetate, leaf ethanol and stem ethyl acetate of D. metel had an LC50 value of <1000 which was included in the toxic category.The LC50 value of flower ethanol reached 121.044 ppm, flower ethyl acetate 105.89 ppm, leaf ethanol 639.589 ppm and stem ethyl acetate 635.276 ppm.Leaf ethanol extract at a concentration of 1000 ppm showed the highest mortality with a percentage of 60% of the total number of A. salina.The flower ethanol extract showed the highest mortality at a concentration of 1000 ppm with a percentage of 70% of the total number of A. salina.Meanwhile, extracts of ethyl acetate from flowers and stems at a concentration of 1000 ppm can cause 100% mortality of A. salina in the first 6 hours.This indicates that the ethyl acetate extract of flowers and stems at a concentration of 1000 ppm has the highest toxic level compared to other concentrations.The LC50 value of the stem ethanol extract and leaf ethyl acetate was >1000 ppm with the stem ethanol value 3905.27ppm and the leaf ethyl acetate 2040.887ppm.The highest mortality in stem ethanol extract and leaf ethyl acetate occurred at a concentration of 1000 ppm with a percentage of 44% of the total amount of A. salina.At the highest concentration it could not kill up to 50% of the tested A. salina samples, this indicates that the stem ethanol extract and leaf ethyl acetate extract are included in the non-toxic category.An extract is categorized as non-toxic if it has an LC50 value >1000 ppm, is categorized as toxic if it has an LC50 value <1000 ppm and is categorized as very toxic if the LC50 value <30 ppm (Meyer et al., 1982).
The high toxicity found in plant extracts can be caused by the content of secondary metabolites of alkaloids, tannins, steroids, and triterpenoids.The most abundant alkaloid content found in D. metel was scopolamine and atropine, with variations in the content of stems, leaves, and flowers ranging from 0.001 to 0.66 μg/mL for scopolamine and 0.001 to 0.27 μg/mL to atropine (Sharma et al., 2021).The compounds contained in these plants can kill A. salina by acting as stomach poisoning.Therefore, if these compounds enter the body of A. salina, the digestive system will be disturbed.In addition, the metabolites present in these plants will also inhibit receptors in the mouth of A. salina.This can result in A. salina not being able to stimulate the taste, so it is unable to recognize its food and causing A. salina to starve to death (Noviati et al., 2012).The content of secondary metabolites in D. metel can have a variety of beneficial biological activities but can be toxic when administered in large quantities (Cinelli and Jones, 2021).Several studies reported damage to the kidney epithelial tissue of rats given ethanol extract of leaves, seeds, and fruit of D. meter (Imo et al., 2018), and significant changes in blood biochemical parameters in rats given methanol, water, and diethyl ether extract of seeds D. stramonium for 14 days (Ogunmoyole et al., 2019).The toxic effect of the aqueous extract of D. metel leaves has also been investigated to cause changes in the pathophysiological conditions of the gills and digestive tract of Cyprinus carpio (Tasneem et al., 2016).
The effects caused by toxic secondary metabolites occur very quickly in just 24 hours and can cause 50% of deaths from A. salina (Rohmah et al., 2019), even reaching 100% at a concentration of 1000 ppm (Al-Hadhrami et al., 2016).Research conducted by Jihad et al. (2019) reported a 75% mortality rate of A. salina after exposure to D. stremonium extract for 24 hours.The toxicity of secondary metabolites found in plants is used for self-defense against predators.This selfdefense mechanism occurs by protecting target organs or inhibiting cell division affected by pathogens (Cutler and Cutler, 2000), or by interfering with the nervous system, membrane transport system, protein synthesis and enzymatic activity from predators (Adibah and Azzreena, 2019).

Figure 1 .
Figure 1.The survival rate of A. salina exposed to (A) ethanol and (B) ethyl acetate extracts of D. metel leaves.

Figure 2 .Figure 3 .
Figure 2. The survival rate of A. salina exposed to (A) ethanol and (B) ethyl acetate extracts of D. metel stems.

Table 1 .
Bioactive compounds of ethanol extracts determined by GC-MS Analysis.

Table 2 .
Bioactive compounds of ethyl acetate extracts determined by GC-MS Analysis.

Table 3 .
LC50 values in A. salina treated with ethanol and ethyl acetate extracts of leaves, stems and flowers of D. metel after 24 hours of observation.